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International Journal of Nanomedicine 2012:7 1115–1125
International Journal of Nanomedicine
Improved oral bioavailability of poorly water-soluble indirubin by a supersaturatable self-microemulsifying drug delivery system
Zhi-Qiang ChenYing LiuJi-Hui ZhaoLan WangNian-Ping FengSchool of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China
Correspondence: Nian-Ping Feng Department of Pharmaceutics, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Zhangjiang Hi-Tech Park, Pudong New District, Shanghai 201203, People’s Republic of China Tel +86 21 5132 2198 Fax +86 21 5132 2198 Email npfeng@hotmail.com
Background: Indirubin, isolated from the leaves of the Chinese herb Isatis tinctoria L, is a
protein kinase inhibitor and promising antitumor agent. However, the poor water solubility of
indirubin has limited its application. In this study, a supersaturatable self-microemulsifying
drug delivery system (S-SMEDDS) was developed to improve the oral bioavailability of
indirubin.
Methods: A prototype S-SMEDDS was designed using solubility studies and phase diagram
construction. Precipitation inhibitors were selected from hydrophilic polymers according to their
crystallization-inhibiting capacity through in vitro precipitation tests. In vitro release of indirubin
from S-SMEDDS was examined to investigate its likely release behavior in vivo. The in vivo
bioavailability of indirubin from S-SMEDDS and from SMEDDS was compared in rats.
Results: The prototype formulation of S-SMEDDS comprised Maisine™ 35-1:Cremophor®
EL:Transcutol® P (15:40:45, w/w/w). Polyvinylpyrrolidone K17, a hydrophilic polymer, was
used as a precipitation inhibitor based on its better crystallization-inhibiting capacity compared
with polyethylene glycol 4000 and hydroxypropyl methylcellulose. In vitro release analysis
showed more rapid drug release from S-SMEDDS than from SMEDDS. In vivo bioavailabil-
ity analysis in rats indicated that improved oral absorption was achieved and that the relative
bioavailability of S-SMEDDS was 129.5% compared with SMEDDS.
Conclusion: The novel S-SMEDDS developed in this study increased the dissolution rate and
improved the oral bioavailability of indirubin in rats. The results suggest that S-SMEDDS is a
superior means of oral delivery of indirubin.
Keywords: supersaturatable self-microemulsifying drug delivery system, indirubin,
bioavailability, oral drug delivery, hydrophilic polymer
IntroductionIndirubin (Figure 1) is a bisindole compound and protein kinase inhibitor, which is
isolated from the leaves of the Chinese herb, Isatis tinctoria L. The drug has various
pharmacological effects, including antitumor and anti-inflammatory activity.1–3 In the
clinical setting, it has been shown to be effective in the treatment of chronic myelo-
cytic leukemia. In addition, indirubin has been shown to have marked antiproliferative
activity and to be a strong inducer of apoptosis in multiple tumor cell types, including
cervical cancer, liver cancer, and lymphoma cell lines.4 The reported mechanisms for
the antitumor effect involve inhibition of DNA and protein synthesis and inhibition of
key protein kinases.5 Long-term animal studies of indirubin have detected neither bone
marrow toxicity nor hematotoxicity.6,7 However, indirubin has poor water solubility,
resulting in low oral bioavailability. Moreover, ingestion often leads to irritation in the
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International Journal of Nanomedicine 2012:7
gastrointestinal tract. Thus, although indirubin is a promising
antitumor agent, its clinical use has been limited.8
Absorption of indirubin is reported to occur by passive
diffusion. The transport of indirubin through the rat intestine
membrane was not influenced by P-glycoprotein or by the
multidrug resistance-associated protein.9,10 From the point of
view of drug concentration, regardless of other issues, such
as the size of the drug molecule and the absorptive surface
area, the higher the drug concentration at the absorption
site, the higher the fraction of absorbed drug transported
by passive diffusion. Improved solubility and a higher dis-
solution rate for poorly water soluble drugs are key factors
to enhance drug absorption.11 Thus, it is considered that
improvement of indirubin bioavailability may be achieved
by improving its solubilization in the gastrointestinal tract
and increasing its dissolution rate in aqueous solution. Recent
studies have focused on the development and evaluation
of a self-emulsifying drug delivery system (SEDDS) for
indirubin,12,13 in which indirubin has a 1.57-fold improve-
ment in bioavailability compared with commercial indirubin
tablets in beagle dogs.
Over the last two decades, self-(micro)emulsifying drug
delivery systems (SMEDDS) have been widely investigated
as an effective means of delivering poorly water-soluble
drugs via the oral route. The system is composed of oil, sur-
factant, and cosurfactant. An emulsion or microemulsion can
be formed upon dispersion of SMEDDS in aqueous solution.
However, when a drug is released from a microemulsion,
precipitation often occurs due to decreased solubility,
leading to decreased drug dissolution and absorption in vivo.
In addition, high amounts of surfactant and cosurfactant can
generate side effects, including irritation of the gastrointes-
tinal tract, changes in intestinal membrane permeability, and
toxicity.14,15 Nevertheless, significant amounts of surfactant
and cosurfactant are required to deliver drugs in dissolved
form. Thus, inhibiting drug precipitation upon mixing
SMEDDS with aqueous solution is a key consideration in
designing these formulations.
A supersaturation process can maintain drug solubiliza-
tion above equilibrium solubility without precipitation.16–19
A high energy form of the drug (in comparison with crystal-
line powder) in solution yielded a supersaturated state with
increased chemical potential. Thus, it is a thermodynamically
unstable system. When a supersaturated drug delivery system
exists at the absorption site for a sufficient period of time, the
higher drug concentration generated from the supersaturated
state may enhance drug absorption.20 Hydrophilic polymers
such as hydroxypropyl methylcellulose (HPMC) and polyvi-
nylpyrrolidone (PVP) can be used in SMEDDS formulations
as precipitation inhibitors to form supersaturatable self-
(micro)emulsifying drug delivery systems (S-SMEDDS).
When S-SMEDDS come into contact with the aqueous
environment of the gastrointestinal tract, the preparations
are first emulsified, and an emulsion or microemulsion is
formed immediately. The drug may be dissolved in free
form or incorporated in emulsion or microemulsion droplets.
Precipitation inhibitors may increase the solubility of the
free drug or the drug in microemulsion and further increase
the concentration gradient of the drug across the intestinal
membrane, which may significantly improve the water solu-
bility of the drug and enhance oral absorption. Hence, more
recently, S-SMEDDS have been developed to improve oral
absorption of poorly water-soluble drugs.21–26 Generation
of drug supersaturation in the gastrointestinal tract seems a
promising way to improve the oral bioavailability of poorly
water-soluble drugs.
The aim of this study was to develop an S-SMEDDS to
improve the oral bioavailability of indirubin. First, proto-
type S-SMEDDS formulations were prepared. Hydrophilic
polymers PVP, HPMC, and polyethylene glycol (PEG) 4000
were used as precipitation inhibitors, and their crystallization-
inhibiting capacities were evaluated. The mechanism of
inhibition of crystallization by the precipitation inhibitors
was explored. Preparations were then assessed based on drug
release and bioavailability.
Materials and methodsMaterialsIndirubin was obtained from Nanjing Zelang Medical
Technology Co Ltd, Nanjing, China. Glyceryl monoli-
noleate (Maisine™ 35-1), diethylene glycol monoethyl
ether ( Transcutol® P), linoleoyl polyoxyl-6 glycerides
( Labrafil® M2125CS), medium-chain triglycerides (Labra-
f ac™ Lipophi le WL1349) and capr y locaproyl
H
O
NH
HN
Figure 1 Chemical structure of indirubin.
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macrogol-8 glycerides ( Labrasol®) were donated by
Gattefossè, Gennevilliers, France. PEG 400, PEG 200,
PEG 4000, and isopropyl myristate were purchased from
Shanghai Chemical Reagent Corporation, Shanghai,
China. Olive oil was sourced from Sictia, Marseille,
France. Caprylic/capric triglyceride (GTCC) was obtained
from Guangdong Indwell Industrial and Trading Co,
Ltd, Guangdong, China. Polyoxyl 40 hydrogenated cas-
tor oil (Cremophor® RH 40), polyoxyl castor oil (Cre-
mophor EL), and povidone K17 (PVP K17) were from
BASF, Ludwigshafen, Germany. Methocel E6 premium
HPMC was purchased from Colorcon, Shanghai, China.
The other chemicals were of analytical grade.
Solubility studiesThe solubility of indirubin in various oils, surfactants, and
cosurfactants was determined as follows. Vehicle (1 g) was
added to capped tubes containing an excess of indirubin.
After the test tubes were sealed, the mixtures in the test
tubes were shaken for 72 hours at 100 strokes per minutes
in a water bath maintained at 25°C, and the tubes were
then centrifuged at 6700 g for 5 minutes. The supernatant
was dissolved in ethyl acetate in a volumetric flask, and
methanol was added to make up the volume. After filtra-
tion through a 0.45 µm membrane filter, the concentration
of indirubin was determined by HPLC. HPLC analysis was
carried out using a LC-2010A HT liquid chromatography
system (Shimadzu Corporation, Kyoto, Japan) as follows:
Kromasil®-5C18 column (250 mm × 4.6 mm, 5 µm); mobile
phase of methanol:water:phosphoric acid (90:10:0.1, v/v/v);
column temperature of 40°C; detection wavelength 225 nm;
flow rate of 1 mL/minute.
Phase diagram constructionThe ternary phase diagrams of mixtures of oil, surfactant, and
cosurfactant at certain ratios were constructed as previously
described.27 After dilution and gentle agitation of the mixtures
in simulated gastric fluid, the emulsification behavior was
observed. The boundaries of the self-microemulsification
regions in the phase diagrams were determined by connecting
the points representing formation of the microemulsion.
Preparation of SMEDDSThe compounds used in the indirubin SMEDDS formulations
are shown in Table 1. Indirubin were prepared by dissolving
the drug in the mixture of Transcutol P, Cremophor EL, and
Maisine 35-1 with continuous stirring. SMEDDS was formed
when the dispersion became transparent. PVP K17 was then
introduced into the SMEDDS preparation and dispersed by
vortexing.
Determination of droplet size and zeta potentialFormulations were diluted in distilled water before analysis.
Droplet size and distribution was measured by photocorrela-
tion spectroscopy using a Nicomp 380/ZLS system (PSS, Port
Richey, FL). Each sample was analyzed in triplicate. Zeta
potential was determined in the same manner.
In vitro evaluation of precipitationTo investigate the precipitation-inhibiting capacity of hydro-
philic polymers, in vitro precipitation assays were conducted
using a RC 806 dissolution tester (Tianjin Tianda Tianfa
Technology Co, Ltd, Tianjin, China) with 250 mL round
vessels at 37°C ± 0.5°C and a stirring speed of 100 rpm. The
formulations including SMEDDS and SMEDDS containing
various hydrophilic polymers were mixed with simulated
gastric fluid to a total volume of 100 mL. At predetermined
times, 0.5 mL of sample was withdrawn and centrifuged at
13,000 rpm for 3 minutes. Supernatant (0.2 mL) was mixed
with 0.4 mL of methanol, and the concentration of indirubin
was analyzed by HPLC.
In vitro releaseIn vitro release assays were carried out according to the
release test method described in the Chinese Pharmacopeia
(2010 edition, small cup method).28 Indirubin S-SMEDDS
(0.5 g) was added to a size zero capsule. The capsule was
then put into the basket in 100 mL of simulated gastric fluid.
Samples (0.5 mL) were removed at 5, 10, 20, 30, 40, 60,
120, 180, 240, and 420 minutes after addition of drug, and
the volume was replaced by adding 0.5 mL of fresh medium.
Table 1 Solubility of indirubin in various vehicles at 25°C
Vehicles Solubility (μmoL/g)
Oils Olive oil 0.145IPM 0.087GTCC 0.154Labrafil M2125CS 0.338Labrafac Lipophile WL1349 0.159Maisine 35-1 0.755
Surfactants Cremophor RH40 0.631Cremophor EL 1.305
Cosurfactants Transcutol P 1.356PEG 400 0.143PEG 200 0.093
Abbreviations: PEG, polyethylene glycol; GTCC, caprylic/capric triglyceride; IPM, isopropyl myristate.
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Improved oral bioavailability of indirubin by S-SMEDDS
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The indirubin concentration was measured as described
above. The cumulative released percentage was calculated
as shown in Equation 1:
Q
C V C V
W
t ti
n
a
=× + ×
×−
=
−
∑ 11
1
100% (1)
in which Q is the cumulative percentage released; Ct is the
indirubin concentration at time t; V represents the volume of
release medium (V = 100 mL); Va is the volume of sampling
aliquot (Va = 0.5 mL); and W is the initial amount of indirubin
in the capsule.
Bioavailability studyThe animal experiments were approved by the institu-
tional animal ethics committee of Shanghai University
of Traditional Chinese Medicine. Male Sprague-Dawley
rats (weight 200 ± 20 g) were divided into three groups
of six animals. Prior to drug administration, the animals
were fasted for 12 hours with free access to water. The
tested preparations were SMEDDS, S-SMEDDS, and
the suspensions. SMEDDS was prepared using the same
method as described for preparation of S-SMEDDS but with
PVP K17 omitted. Suspensions were prepared by dispers-
ing indirubin in sodium carboxymethyl cellulose solution
with glycerin as a wetting agent. Rats were administered
a indirubin dose of 2.58 mg/kg for each preparation by
gavage, followed by 1 mL of physiological saline. Blood
samples (0.5 mL) were collected into heparinized tubes
via retro-orbital puncture at hours 0.5, 1, 2, 3, 4, 5, 6, 8,
10, 12, and 24 post dose. Blood samples were centrifuged
at 2000 g for 5 minutes. Plasma was kept at −20°C until
analysis, as described below.
A 0.2 mL sample of plasma was mixed with an equal
volume of saturated ammonium sulfate solution and vortexed
for 10 seconds. Ethyl acetate (0.6 mL) was added, and the
mixture was vortexed for 3 minutes. After centrifugation at
10,000 rpm for 3 minutes, the upper layer was removed and
dried under gentle flowing nitrogen. Residuals were reconsti-
tuted in 100 µL of methanol. The mixture was vortexed for
20 seconds and then centrifuged at 13,000 rpm for 3 minutes.
The supernatant was used for content analysis.
Indirubin concentrations in rat plasma were determined
using a Waters Acquity UPLC-Micromass ZQ 2000 system
(Waters Corporation, Milford, MA). An Acquity UPLC
R BEH C18 column (1.7 µm, 2.1 × 50 mm) was used at
30°C. The mobile phase A was 0.1% formic acid in water.
The mobile phase B was methanol. The gradient program was
set as follows: 0–6 minutes: 40% A, 60% B; 6–7 minutes:
10% A, 90% B; 7–9 minutes: 40% A, 60% B. The flow rate
of the mobile phase was kept at 0.3 mL/minute. The injection
volume was 10 µL. Triple-quadruple tandem mass spectro-
metric detection was operated in positive ionization mode
with m/z of 262.3, a source temperature of 120°C, capillary
voltage of 3000 V, desolvation flow rate of 500 L/hour, and
desolvation temperature of 250°C.
Data analysisPharmacokinetic parameters were obtained using DAS
2.0 software. Statistical analysis for the pharmacokinetic
parameters was carried out using one-way analysis of vari-
ance. A P value of ,0.05 was considered to be statistically
significant.
Results and discussionSMEDDS formulationInitially, SMEDDS were developed as prototypes of
S-SMEDDS. To obtain a SMEDDS formulation, we deter-
mined the solubility of indirubin in various oils, surfactants,
and cosurfactants. The results are presented in Table 1. The
solubility of indirubin in the tested oils, in decreasing order,
was as follows: Maisine 35-1 . Labrafil M2125CS .
Labrafac Lipophile WL1349 . GTCC . olive oil .
isopropyl myristate. Solubility of indirubin in Cremophor
EL (surfactant) and Transcutol P (cosurfactant) was mark-
edly higher than in other vehicles, indicating superior dis-
solving capability for indirubin. Based on these results, we
selected Maisine 35-1 for the oil phase, Cremophor EL as the
surfactant, and Transcutol P as the cosurfactant for further
investigation.
A phase diagram was constructed to identify the self-
microemulsification regions. A series of formulations
containing Maisine 35-1, Cremophor EL, and Transcutol
P were prepared and diluted 200-fold in simulated gastric
fluid at 37°C. A dispersion with a transparent appearance
was regarded as a microemulsion. The phase diagram is
shown in Figure 2.
Based on analysis of self-microemulsifying regions,
three formulations were selected for further assessment of
droplet size, polydispersibility, and equilibrium solubility.
As shown in Table 2, the droplet size of formulation C was
clearly smaller than that of the other formulations. In addi-
tion, formulation C had better solubilizing capacity than
the other formulations. In a previous study, we found that
both the oil percentage and surfactant to cosurfactant ratio
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International Journal of Nanomedicine 2012:7
influence equilibrium solubility.29 As shown in Table 1, the
Transcutol P cosurfactant had better dissolving capability
for indirubin than Cremophor EL. This may explain the fact
that when the oil percentage was the same, formulation C
(with higher Transcutol P content) exhibited better drug
solubility than the other two formulations. Based on these
findings, formulation C was selected as the final prototype
S-SMEDDS.
Screening for a precipitation inhibitorA hydrophilic polymer is an essential excipient in a
S-SMEDDS formulation. Hydrophilic polymers, such as
PVP, HPMC, and PEG, have been found to be useful as
precipitation inhibitors.18,30–34 In this study, HPMC, PEG
4000, and PVP K17 were assayed for inhibition of crystal-
lization in vitro.
As reported previously, evaluation of the true free drug
concentration in preparations dispersing in aqueous sys-
tems is difficult, mainly due to the complex drug state and
dynamic processes during dispersion.21–23 When SMEDDS
is dispersed into aqueous solution, the drug may exist in
three main states, namely, as a free molecule in solution,
solubilized in microemulsion or similar vehicles, and/or
precipitated as solid particles. To evaluate drug precipitation
behavior accurately, it is important to separate precipitated
drug from the dispersion system upon mixing SMEDDS with
an aqueous solution. Syringe filters method and centrifuga-
tion method are generally used methods.24,25,35 The syringe
filter method may separate a solution with precipitated drugs
by filtration, and the centrifugal method achieves separation
by centrifugation. In addition, Gao et al reported an improved
approach using focused beam reflectance measurement
technology.23 Their method is a fast and convenient in situ
measurement technology that may detect particles within
the size range of 1–1000 µm.23 However, it was noted
that focused beam reflectance measurement cannot detect
particles less than 1 µm in size. In this study, we compared
membrane filtration and centrifugation by evaluating the
recovery and repeatability of each method. We found that
the centrifugation method yielded higher recovery and
repeatability than filtration, perhaps by eliminating loss by
adsorption to the filter (data not shown). It should also be
noted that in addition to free molecules in solution and solu-
bilized drug in microemulsion or other vehicles, nanosized
solid drug particles may also be present in supernatants
after centrifugation.
The indirubin concentrations in simulated gastric fluid
after dilution of SMEDDS (without hydrophilic polymers)
and S-SMEDDS formulations are shown in Figure 3.
Figure 3A shows the indirubin concentration-time profile
using varying amounts of PEG 4000 as a precipitating
inhibitor. The results indicate that the formulation with 2%
PEG 4000 achieved the highest drug concentration compared
with the other formulations. PEG 4000 was finely dissolved
in SMEDDS, but as the amount of PEG 4000 increased, the
dispersions became increasingly viscous. At 5% PEG 4000,
the dispersions became gel-like due to high viscosity, which
may hinder self-microemulsification.
In the case of HPMC, compared with HPMC-free
SMEDDS, there was no marked enhancement of drug
release in the presence of 0.5%, 2%, and 5% HPMC
(Figure 3B). This result indicated that HPMC appeared
1.00
0.75
0.50
0.25
0.001.000.750.500.25
Masine 35-1
0.00
1.00
0.75
0.50
0.25
0.00
Cre
mop
hor E
L Transcutol P
Figure 2 Ternary system containing Maisine 35-1/Cremophor EL/Transcutol P. Region of efficient self-microemulsification is bound by the solid line, and the filled circles represent compositions that were evaluated.
Table 2 Droplet size, polydispersibility, and solubility for the three tested SMEDDS formulations
Formulation composition (Maisine 35-1:Cremophor EL:Transcutol P, w:w:w)
Droplet size (nm) Polydispersibility Solubility (μg/g)
A 15:50:35 93.62 0.284 424.3B 15:30:55 91.38 0.524 437.9C 15:40:45 22.17 0.193 474.8
Abbreviation: SMEDDS, self-microemulsifying drug delivery system.
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Improved oral bioavailability of indirubin by S-SMEDDS
International Journal of Nanomedicine 2012:7
less effective as a precipitation inhibitor. As shown in
Figure 3C, the highest release was achieved when 0.5%
PVP K17 was used. In addition, PVP K17 was well
dispersed in SMEDDS, and the addition of PVP K17 to
SMEDDS had no significant influence on the viscosity of
the dispersions. Based on these results, we concluded that
the precipitation-inhibiting capacity of the three hydro-
philic polymers was in the order PVP K17 . PEG 4000 .
HPMC, and that 0.5% PVP K17 can efficiently retard
precipitation and maintain a higher indirubin concentra-
tion for approximately 2 hours or longer. Therefore, 0.5%
of PVP K17 was selected to add to SMEDDS. The final
S-SMEDDS formulation composition was Maisine
35-1:Cremophor EL:Transcutol P 15%:40%:45% (w/w/w)
with 0.5% PVP K17.
The indirubin molecule is formed using a π-conjugated
system. There are strong hydrogen bonds between an oxy-
gen atom and adjacent hydrogen atom. Hydrogen bonds are
SMEDDS
1600
1400
1200
1000
8000 50 100
T (min)
A
B
C
Ind
iru
bin
co
nce
ntr
atio
n (
ng
/mL
)In
dir
ub
in c
on
cen
trat
ion
(n
g/m
L)
Ind
iru
bin
co
nce
ntr
atio
n (
ng
/mL
)
T (min)
T (min)
150 200
2000
1800
1600
1400
1200
1000
8000 50 100 150 200
2000
1800
1600
1400
1200
1000
8000 50 100 150 200
2000
1800
SMEDDS+5% PEG 4000
SMEDDS+2% PEG 4000
SMEDDS+0.5% PEG 4000
SMEDDS
SMEDDS+5% HPMC
SMEDDS+2% HPMC
SMEDDS+0.5% HPMC
SMEDDS
SMEDDS+5% PVP K17
SMEDDS+2% PVP K17
SMEDDS+0.5% PVP K17
Figure 3 In vitro indirubin concentration-time profiles from the SMEDDS formulations and S-SMEDDS with hydrophilic polymers of (A) PEG 4000, (B) HPMC, and (C) PVP K17 (n = 5).Abbreviations: SMEDDS, self-microemulsifying drug delivery system; S-SMEDDS, supersaturatable self-microemulsifying drug delivery system; PEG, polyethylene glycol; PVP, polyvinylpyrrolidone; HPMC, hydroxypropyl methylcellulose.
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responsible for the stability of the molecule. One hexatomic
ring can be formed by intramolecular hydrogen bonds,
and therefore lower the ring strain and the molecular total
system energy.36 Therefore, the indirubin molecule is stable
and has low water solubility. We found that both PEG 4000
and indirubin were well dissolved in SMEDDS. We propose
that when each PEG 4000 molecule (Figure 4A) opened the
two parallel helical bonds, the indirubin molecules were
incorporated into the coiling chain to cause molecular
dispersion. Meanwhile, the intramolecular hydrogen bond
was activated upon dissolving of indirubin in SMEDDS,
and exchanging of a hydrogen bond with PEG 4000 may
occur (Figure 4D). In a previous report, it was proposed
that the adsorption of the polymer onto the crystal surface
may play a role in inhibition of crystallization, caused by
a hydrogen bond between the drug and the hydrophilic
polymer.37–39 Therefore, from this perspective, the hydrogen
bonds between indirubin and PEG 4000 may contribute
R1
O
O
O
O
O
O
O
OO
H
H
H
CH
H
HN
N
N
N
N
D
E
C
B
A
H OH
HN
R2
H2C CH2HC
R3
R3
CH3
CH3R=H oror
Hx
O
O
CCH2H
HO
OH
Nn
n
OR
OR
O O
O
O
OR
OR
OR
OR
R
nOR
Figure 4 Molecular structures of (A) PEG 4000, (B) PVP, (C) HPMC and schematic diagram of hydrogen bonds between indirubin, (D) PEG 4000, and (E) PVP.Abbreviations: PEG, polyethylene glycol; PVP, polyvinylpyrrolidone; HPMC, hydroxypropyl methylcellulose.
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Improved oral bioavailability of indirubin by S-SMEDDS
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80
100
60
40
20
00 50 100 150 200
Time (min)
Cu
mu
lati
ve p
erce
nta
ge
rele
ased
(%
)
250 300 350 400 450
SMEDDS
S-SMEDDS
Figure 5 Cumulative release profile of indirubin SMEDDS and S-SMEDDS.Abbreviations: SMEDDS, self-microemulsifying drug delivery system; S-SMEDDS, supersaturatable self-microemulsifying drug delivery system.
to the retarded precipitation of indirubin after dilution of
SMEDDS.
Another factor responsible for the role of the precipita-
tion inhibitor is thought to be the incorporation of drug
molecules into polymer aggregates.40 In the case of PVP K17
(Figure 4B), after PVP K17 and indirubin were dissolved
in SMEDDS, PVP K17 showed reticulate structures, and
indirubin molecules may be incorporated into the reticulate
structures resulting in molecular dispersion. Indirubin-PVP
K17 hydrogen bonds were formed between the carbonyl
group of the pyrrolidone ring of PVP K17 and the amino
group in the pyrrole ring as well as the amido hydrogen of
indirubin (Figure 4E). Hydrogen bonding and incorporation
of indirubin into reticulate structures, as well as high viscos-
ity, may efficiently inhibit the aggregation and crystallization
of indirubin. Further study of the indirubin-PVP K17 interac-
tion using infrared spectroscopy or other analytic strategies
may clarify these matters.
HPMC (Figure 4C), a cellulosic polymer, has been
reported to be an efficient crystal growth inhibitor for many
drugs in aqueous dispersions.18,33,39 Approximately 60% of
the hydroxyl groups in HPMC are not substituted. HPMC
can thus form both intramolecular and intermolecular
hydrogen bonds. Thus, HPMC is more hydrophobic than
PVP K17 and PEG 4000. Once the hydrogen bonds are
disrupted in the dissolved state, HPMC may interact with a
drug and form hydrogen bonds, which is likely to retard drug
precipitation. Furthermore, based on the molecular struc-
ture, HPMC may provide more hydrogen bonding groups
per monomer unit compared with PVP K17. (Interaction
of indirubin and HPMC is not described herein due to the
complex substituent group leading to uncertain hydrogen
bond formation). However, in this study, we found that
HPMC cannot be dissolved in indirubin SMEDDS. Thus,
it appears that HPMC exerted no precipitation-inhibiting
action on indirubin SMEDDS.
In vitro release studyThe in vitro release assay (Figure 5) showed that 70% of
the drugs was released from S-SMEDDS in 60 minutes,
while approximately 50% of the drugs was released from
SMEDDS in 120 minutes. S-SMEDDS exhibited faster
release behavior than SMEDDS. However, the percentage
released in the case of S-SMEDDS decreased slightly after
60 minutes, and decreased slowly to approximately 60% at
240 minutes. The released drug was detected in the dissolved
form either in solution or in the microemulsion-like vehicles.
Thus, the decrease in the amount of dissolved drug indicated
that precipitation had occurred. A supersaturatable system
is thermodynamically unstable, and a supersaturatable drug
solution has a tendency to return to its equilibrium state via
drug precipitation.20 Thus, the supersaturation process may
only keep the drug dissolved for a limited period of time.
With increasing time, drug precipitation may occur, which
results in a decreased amount of dissolved drug. Because
there is no supersaturation process involved in SMEDDS,
the percentage of drug released increased steadily with the
passage of time. Almost the same percentage of drug release
was achieved for the two preparations at 420 minutes. Based
on the results of our in vitro release study, we surmise that
S-SMEDDS would have higher bioavailability in vivo than
SMEDDS.
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Bioavailability studyUltra performance liquid chromatography/mass spectrom-
etry chromatograms are shown in Figure 6, which indi-
cate that endogenous substances in plasma did not affect
detection of indirubin. Figure 7 shows the mean plasma
concentration profile of indirubin from the suspension,
SMEDDS, and S-SMEDDS after oral administration to
rats. The plasma concentration of indirubin after dosing of
the indirubin suspension could not be detected due to poor
oral absorption in vivo. S-SMEDDS containing indirubin
1.51 1.69
2.38
2.99
3.36
Time
Time
4.003.803.603.403.203.002.802.602.402.202.001.801.601.401.20
4.003.803.603.403.203.002.802.602.402.202.001.801.601.401.20
4.00Time
3.803.603.403.203.002.802.602.402.202.001.801.601.401.20
0
%
100
0
%
1.05
1.21
1.42
1.64 1.78 1.92
2.492.63 2.822.92
3.073.45
3.69
3.24
2.34
3.783.95
3.81
100
0
%
100 2.36
3.151.50
A
B
C
3.15
Figure 6 Ultra performance liquid chromatography/mass spectrometry chromatograms of (A) blank plasma, (B) blank plasma spiked with indirubin, and (C) plasma sample after oral administration of indirubin to a rat.
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Improved oral bioavailability of indirubin by S-SMEDDS
International Journal of Nanomedicine 2012:7
provided higher plasma drug concentrations than SMEDDS
containing indirubin.
Mean area under the curve (AUC), peak plasma
concentration, and relative bioavailability are listed in
Table 3. Parameters for the suspensions were not analyzed
because the plasma concentration could not be detected.
The relative bioavailability was calculated by dividing
AUC0–24 h
(S-SMEDDS) by AUC0–24 h
(SMEDDS). The
peak plasma concentration and mean residence time values
for indirubin from S-SMEDDS were not significantly dif-
ferent than those from SMEDDS. However, the AUC for
S-SMEDDS was significant larger than that of SMEDDS
(P , 0.05).
These results suggest that S-SMEDDS might be useful
for enhancing the oral absorption of indirubin. Furthermore,
the results of the in vivo bioavailability study appear to cor-
relate with those of the in vitro release study, which indicates
a faster release rate from S-SMEDDS than from SMEDDS.
For poorly water soluble drugs, dissolution is generally the
rate-limiting step to drug absorption. A higher release rate
might play an important role in enhancing bioavailability. In
addition, S-SMEDDS provides indirubin in supersaturated
conditions upon mixing with aqueous dispersions, allowing
indirubin to dissolve rather than precipitate, and further
enhancing bioavailability.
ConclusionThe novel S-SMEDDS developed in this study increased
the dissolution rate and improved the oral absorption of
indirubin. The S-SMEDDS is a promising way to deliver
indirubin by the oral route.
AcknowledgmentsThis work was financially supported by a grant (J50302) from
the Shanghai Education Committee, a grant (10XD14303900)
from the Science and Technology Commission of Shanghai
Municipality, and by grants (NCET08-0898 and IRT1071) from
the State Education Ministry, People’s Republic of China.
DisclosureThe authors report no conflicts of interest in this work.
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90
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10
0
Ind
iru
bin
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(n
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Cmax (ng/mL)
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